Cholesteryl ester transfer protein (cetp) inhibition in the treatment of cancer

ABSTRACT

In one embodiment, the invention provides methods of treatment which use therapeutically effective amounts of Cholesteryl Ester Transfer Protein (CETP) inhibitors to treat a variety of cancers. In certain embodiments, the inhibitor is a CETP-inhibiting small molecule, CETP-inhibiting antisense oligonucleotide, CETP-inhibiting siRNA or a CETP-inhibiting antibody. Related pharmaceutical compositions, kits, diagnostics and screens are also provided.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication No. 62/120,100, filed Feb. 24, 2015, entitled “CholesterylEster Transfer Protein (CETP) Inhibition in the Treatment of Cancer”,the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

In one embodiment, the invention provides methods of treatment which usetherapeutically effective amounts of Cholesteryl Ester Transfer Protein(CETP) inhibitors to treat a variety of cancers. In certain embodiments,the inhibitor is a CETP-inhibiting small molecule, CETP-inhibitingantisense oligonucleotide, CETP-inhibiting siRNA or a CETP-inhibitingantibody.

Related pharmaceutical compositions, kits, diagnostics and screens arealso provided.

BACKGROUND OF THE INVENTION

The majority of breast cancers occur in postmenopausal women, with 75%of these tumors being estrogen dependent as defined as estrogen receptor(ER) positive. Tamoxifen, an anti-estrogen, has been the mainstay oftreatment for hormone-dependent breast cancers. However, recent clinicaltrials have shown that inhibitors of aromatase, which catalyze therate-limiting step of estrogen biosynthesis, may be more effective thantamoxifen in treating hormone-dependent breast cancers in postmenopausalwomen. Unfortunately, resistance to both these endocrine therapies isinevitable in metastatic breast cancer.

We have screened derivatives of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) against various human cancer cell lines and we havedetermined that a derivative acetyl plumbagin (“AP”) is active in breastcancer cell lines models. Further, we have shown that AP has severaladvantages over Tamoxifen in these cell line models. See U.S. PatentApplication Document No. 20140107196, which is incorporated herein inits entirety.

There remains a compelling need to develop more effective therapies forbreast cancer patients, particularly those with acquired anti-estrogenresistance, in addition to those with intrinsic resistance toanti-estrogen and anti-HER2 therapies.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method of treating a subjectwho suffers from a cancer, the method comprising administering to thesubject a therapeutically effective amount of one or more compositionsselected from the group consisting of a Cholesteryl Ester TransferProtein (CETP) inhibiting small molecule as described hereinafter, aCETP inhibiting antisense oligonucleotide, a CETP inhibiting siRNA and aCTEP inhibiting antibody.

In another embodiment, the survival time of a patient suffering frombreast cancer is increased by concomitant administration of a CETPinhibitor and one or more additional anticancer agents selected from thegroup consisting of tamoxifen, paclitaxel and fluorouracil (5-FU). Insome embodiments, the subject is co-administered plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone), or an analog, derivative,pharmaceutically acceptable salt, enantiomer, diastereomer, solvate orpolymorph thereof. Methods of treatment of the invention areparticularly useful in the treatment of a patient who suffers from aform of refractory breast cancer, who has developed an acquiredanti-estrogen resistance or who exhibits an intrinsic resistance toanti-estrogen and anti-HER2 therapies.

In still other embodiments, the invention provides a method ofpredicting the responsiveness of a patient suffering from a cancer totreatment with one or more anticancer agents selected from the groupconsisting of tamoxifen, paclitaxel and fluorouracil (5-FU), the methodcomprising measuring levels of CETP in a sample taken from the patientand comparing measured values to control CETP values of a healthysubject, wherein decreased survival times are predicted where measuredCETP levels exceed control levels. Preferably, the measured CETP levelsare often mRNA levels of CETP in cancer cells or plasma CETP levels.

The invention also provides a method of determining whether acomposition is effective in the treatment of one or more cancers, themethod comprising:

(a) contacting a cancer cell sample with the composition;(b) measuring at least one cancer cell sample indicator selected fromthe group consisting of cellular viability, cancer cell levels ofmitochondrial outer membrane potential (MOMP) and cellular levels ofapoptosis-associated proteins; and(c) comparing measured cancer cell sample viability, MOMP levels and/orlevels of apoptosis-associated proteins with cell viability, MOMP levelsand/or levels of apoptosis-associated proteins in a control cancer cellsample which is contacted with plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone), or an analog, derivative,pharmaceutically acceptable salt, enantiomer, diastereomer, solvate orpolymorph thereof;wherein the composition is determined to be effective in the treatmentof one or more cancers if measured levels of cell viability, MOMP and/orapoptosis-associated proteins are approximately the same as or less thancomparable control levels.

Another screening method of the invention involves determining whether acomposition is effective in the treatment of one or more cancers, themethod comprising:

(a) contacting a cancer cell sample with the composition;(b) measuring cellular Cholesteryl Ester Transfer Protein (CETP) levels;and(c) comparing measured cancer cell CETP levels with CTEP levels of acontrol cancer cell sample which is contacted with plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone), or an analog, derivative,pharmaceutically acceptable salt, enantiomer, diastereomer, solvate orpolymorph thereof;wherein the composition is determined to be effective in the treatmentof one or more cancers if measured CTEP levels are approximately thesame as or less than comparable control levels.

Significantly, we have discovered that acetyl plumbagin (AP) and relatedderivatives are CETP-inhibitors that, like other CETP inhibitors such asthose described hereinafter, can be combined with Tamoxifen to increasethe viability of normal cells and significantly decrease the viabilityof cancer cells. CETP inhibitor co-therapy protects normal cells fromtoxic effects of Tamoxifen and enables tamoxifen to kill cancer cells athalf of the normal recommended dose.

These and other aspects of the invention are described in greater detailin the Detailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: MTT and APOPercentage assays to assess the differentialcytotoxicity and apoptosis inducing potential of AP and PL in MCF-7cells. Viability was determined using MTT assay after incubating theMCF-7 cells with different concentrations of AP and PL (A), at differenttime-points (B). The apoptosis inducing potential of 10 μM AP and PL inMCF-7 was determined by using APOPercentage assay performed at intervalsof 2 and 6 h.

FIG. 2: Mode of induction in MCF-7 cells after treatment with AP and PLwas determined by measuring changes in MOMP and levels ofapoptosis-associated proteins. The cells were incubated with 10 μM APand PL for different time intervals to capture the timeline of apoptosisinduction in MCF-7 cells. The changes in MOMP were measured using flowcytometry after treating MCF-7 cells with PL and AP for 1, 2 and 4 h(A). The alteration in expression of several apoptosis-associatedproteins were determined using western blotting after incubating MCF-7cells for 1, 2, 4, 6 h with PL (C, E) and AP (D. F).

FIG. 3: Comparative efficacy and toxicity study in MCF-7 xenograft nudemouse model of BC. The efficacy of single i.p. injection of vehicle, PL(2 mg/kg) and AP (5 mg/kg) in 5 mice in each group was compared over aperiod of 21 days. The comparison of tumor sizes (A), tumor volume (B)and body weight (C) demonstrated the efficacy of compound AP as comparedto PL. Plasma levels of parameters ALT (D) and AST (E) representingliver toxicity were estimated in vehicle, PL and AP treated mice groupsshowing that AP even reduced vehicle associated toxicity in treatedmice.

FIG. 4: Lipid raft GM1 staining in MCF-7 cells with the Vybrant® AlexaFluor® 488 Lipid Raft Labeling Kit. Cells were cultured in 96-wellplates and stained with CT-B Alexa 488 in untreated cells, and aftertreatment with 10 μM PL or AP for 10 min, 30 min, 1 h and 2 h.

FIG. 5: Cholesterol depletion by PL and AP. BJ, BT20 and MCF-7 cellswere treated with 10 μM PL or AP for 2 h and cholesterol levels weredetermined with the Amplex Red Cholesterol kit as per manufacturer'sinstructions. Cells treated with 5 mg/ml MBCD was used as a positivecontrol and fluorescence was measured with a Tecan plate reader. MCF-7cells were pre-incubated with 1 mM cholesterol for 1 h followed bytreatment with 10 μM PL or AP for 2 h (A). Apoptosis was determined byflow cytometry with the APOPercentage kit or Cells treated with MBCD and5 mM H₂O₂ served as positive controls (B). Values represent the means±SDof three independent experiments.

FIG. 6: Levels of total Cholesterol (A) and unbound free cholesterol (B)in mice serum after treatment with 2 mg/kg PL or 5 mg/kg AP weredetermined with the Amplex Red assay kit. Levels of total cholesterol,HDL and LDL/VLDL in mice serum determined with ABNOVA HDL and LDL/VLDLAssay kit (C). Values represent the means±SD of three independentexperiments. Reference values have been adapted fromhttp://www.abcam.com/hdl-and-ldlvldl-cholesterol-assay-kit-ab65390.html.

FIG. 7: Levels of CETP mRNA in tumor (A) and protein in serum (B) inmice tumor samples after treatment with 2 mg/kg PL or 5 mg/kg AP weredetermined with the RT-PCR and western blotting, respectively. Foldchange in expression of serum protein was determined using densitometryanalysis of western blots using ImageJ (C). CETP activity in serum wasestimated using CETP activity kit (name of kit/manufacturer) (D). Valuesrepresent the means±SD of three independent experiments. * where p≦0.05,and ** where p≦0.005.

FIG. 8: Levels of total cholesterol in MCF-7 control (cntl) siRNA andCETP knock-out cells after treatment with 10 μM PL or AP were determinedwith the Amplex Red assay kit. Values represent the means±SD of threeindependent experiments.

FIG. 9: The effect of CETP mRNA silencing on proliferation of MCF-7cells was measured by counting cells with a Countess cell counter(Invitrogen) over a period of six days. Values represent the means±SD ofthree independent experiments.

FIG. 10: The effect of CETP mRNA silencing on MMP (A), Capase-3/7activity (B) and apoptosis in MCF-7 (C) and BJ (D) untransfected, cntlsiRNA and CETP knockout cells after treatment with 10 μM PL or AP.Values represent the means±SD of three independent experiments.

FIG. 11: Over-expression of CETP mRNA in six published breast cancermicroarray datasets available in Oncomine (website: oncomine.org).

FIG. 12: The effect of CETP mRNA knockout on Capase-3/7 activity (A) andapoptosis (B) in MCF-7 cntl siRNA and CETP knockout cells aftertreatment with 25 μM tamoxifen for 24 h. Values represent the means±SDof three independent experiments.

FIG. 13: The effect of AP on viability of BJ and MCF-7 cells whencombined with different doses of various chemotherapeutic drugs for 24h. Values represent the means±SD of three replicates.

FIG. 14: Illustration of the amount of stabilized CETP protein in thepresence of compounds using TSA. MCF-7 cells were left untreated (Untx)and were treated with 10 μM of AP/PL for two hours. TSA was performedusing temperature range 56° C. to 70° C. with 2° C. intervals. Thewestern blotting was performed after the assay and CETP was detected.The experiment was performed in duplicate.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional chemical synthetic methods and other biological andpharmaceutical techniques within the skill of the art. Such techniquesare well-known and are otherwise explained fully in the literature.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms in which case each carbon atomnumber falling within the range is provided), between the upper andlower limit of that range and any other stated or intervening value inthat stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedherein below, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein. Within its use incontext, the term generally refers to a single compound comprising ahydrophobic moiety and a linker which is capable of reacting and forminga covalent bond with a fusion protein as otherwise described herein. Incertain instances the term may also refer to stereoisomers and/oroptical isomers (including racemic mixtures) or enantiomericallyenriched mixtures of disclosed compounds. Compounds which are disclosedare those which are stable and where a choice of substituents and claimelements is available, the substituent or claim element is chosen suchthat stable compounds are formed from the disclosed elements andsubstituents. The symbol

in a chemical structure or formula signifies that either a double orsingle bond may be present between the atoms to which such symbol isattached, depending upon the valence of those atoms and substituentswhich are on such atoms.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, especially including adomesticated mammal and preferably a human, to whom a treatment orprocedure, including a prophylactic treatment or procedure is performed.For treatment of those infections, conditions or disease states whichare specific for a specific animal such as a human patient, the termpatient refers to that specific animal. In most instances, the patientor subject of the present invention is a domesticated/agriculturalanimal or human patient of either or both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition which, in context, isused to produce or effect an intended result, whether that resultrelates to the treatment of a cancer in a patient or subject. The termeffective subsumes all other effective amount or effective concentrationterms which are otherwise described or used in the present application.

“Hydrocarbon” or “hydrocarbyl” refers to any monovalent (or divalent inthe case of alkylene groups) radical containing carbon and hydrogen,which may be straight, branch-chained or cyclic in nature. Hydrocarbonsinclude linear, branched and cyclic hydrocarbons, including alkylgroups, alkylene groups, saturated and unsaturated hydrocarbon groupsincluding aromatic groups both substituted and unsubstituted, alkenegroups (containing double bonds between two carbon atoms) and alkynegroups (containing triple bonds between two carbon atoms). In certaininstances, the terms substituted alkyl and alkylene are sometimes usedsynonymously.

“Alkyl” refers to a fully saturated monovalent radical containing carbonand hydrogen, and which may be cyclic, branched or a straight chain.Examples of alkyl groups are methyl, ethyl, n-butyl, n-hexyl, n-heptyl,n-octyl, n-nonyl, n-decyl, isopropyl, 2-methyl-propyl, cyclopropyl,cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclopentylethyl,cyclohexylethyl and cyclohexyl. Preferred alkyl groups are C₁-C₆ alkylgroups. “Alkylene” refers to a fully saturated hydrocarbon which isdivalent (may be linear, branched or cyclic) and which is optionallysubstituted. Preferred alkylene groups are C₁-C₆ alkylene groups. Otherterms used to indicate substitutent groups in compounds according to thepresent invention are as conventionally used in the art.

The term “aryl” or “aromatic”, in context, refers to a substituted orunsubstituted monovalent aromatic radical having a single ring (e.g.,benzene or phenyl). Other examples of aryl groups, in context, mayinclude heterocyclic aromatic ring systems “heteroaryl” groups havingone or more nitrogen, oxygen, or sulfur atoms in the ring (5- or6-membered heterocyclic rings) such as imidazole, furyl, pyrrole,pyridyl, furanyl, thiene, thiazole, pyridine, pyrimidine, pyrazine,triazole, oxazole, among others, which may be substituted orunsubstituted as otherwise described herein.

The term “heterocyclic group” “heterocycle” as used throughout thepresent specification refers to an aromatic (“heteroaryl”) ornon-aromatic cyclic group forming the cyclic ring and including at leastone and up to three hetero atoms such as nitrogen, sulfur or oxygenamong the atoms forming the cyclic ring. The heterocyclic ring may besaturated (heterocyclic) or unsaturated (heteroaryl). Exemplaryheterocyclic groups include, for example pyrrolidinyl, piperidinyl,morpholinyl, pyrrole, pyridine, pyridone, pyrimidine, imidazole,thiophene, furan, pyran, thiazole, more preferably pyrimidinyl,pyrrolidinyl, piperidinyl, morpholinyl, oxazole, isoxazole, pyrrole,pyridine, thiophene, thiazole and even more preferably pyrimidinyl,especially uracil or cytosine which are optionally substituted, furyl,3-methylfuryl, thiazole, piperazinyl, N-methylpiperazinyl,tetrahydropyranyl and 1,4-dioxane, among others. Additional heterocyclicgroups include oxazole, benzoxazole, pyrrole, dihydropyrrole,benzopyrrole, benzodihydropyrrole, indole, indolizine, among others.

Exemplary heteroaryl moieties which may be used in the present inventioninclude for example, pyrrole, pyridine, pyridone, pyridazine,pyrimidine, pyrazine, pyrazole, imidazole, triazole, tetrazole,oxadiazole, sulfur-containing aromatic heterocycles such as thiophene;oxygen-containing aromatic heterocycles such as furan and pyran, andincluding aromatic heterocycles comprising 2 or more hetero atomsselected from among nitrogen, sulfur and oxygen, such as thiazole,thiadiazole, isothiazole, isoxazole, furazan and oxazole. Furtherheteroaryl groups may include pyridine, triazine, pyridone, pyrimidine,imidazole, furan, pyran, thiazole. Pyrimidine groups, especially uraciland cytosine, optionally substituted, are preferred.

The term “substituted” shall mean substituted at a carbon (or nitrogen)position within context, hydroxyl, carboxyl, cyano (C≡N), nitro (NO₂),halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl,especially a methyl group such as a trifluoromethyl), alkyl group(preferably, C₁-C₁₀, more preferably, C₁-C₆), alkoxy group (preferably,C₁-C₆ alkyl or aryl, including phenyl and substituted phenyl), ester(preferably, C₁-C₆ alkyl or aryl) including alkylene. ester (such thatattachment is on the alkylene group, rather than at the ester functionwhich is preferably substituted with a C₁-C₆ alkyl or aryl group),preferably, C₁-C₆ alkyl or aryl, halogen (preferably, F or Cl), nitro oramine (including a five- or six-membered cyclic alkylene amine, furtherincluding a C₁-C₆ alkyl amine or C₁-C₆ dialkyl amine which alkyl groupsmay be substituted with one or two hydroxyl groups), amido, which ispreferably substituted with one or two C₁-C₆ alkyl groups (including acarboxamide which is substituted with one or two C₁-C₆ alkyl groups),alkanol (preferably, C₁-C₆ alkyl or aryl), or alkanoic acid (preferably,C₁-C₆ alkyl or aryl). Preferably, the term “substituted” shall meanwithin its context of use alkyl, alkoxy, halogen, ester, keto, nitro,cyano and amine (especially including mono- or di-C₁-C₆ alkylsubstituted amines which may be optionally substituted with one or twohydroxyl groups). Any substitutable position in a compound according tothe present invention may be substituted in the present invention, butno more than 3, more preferably no more than 2 substituents (in someinstances only 1 or no substituents) is present on a ring. Preferably,the term “unsubstituted” shall mean substituted with one or more Hatoms.

“Halogen” or “halo” may be fluoro, chloro, bromo or iodo.

A “hydrolyzable moiety” can be methyl, t-butyl, benzyl, p-methoxybenzyl,p-nitrobenzyl, allyl, trityl, methoxymethyl, 2-methoxypropyl,methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl,tetrahydrothiopyranyl, and trialkylsilyl ethers such as trimethylsilylether, triethylsilyl ether, dimethylarylsilyl ether, triisopropylsilylether and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl,phenylacetyl, formyl, mono-, di-, and trihaloacetyl such aschloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; andcarbonates including but not limited to alkyl carbonates having from oneto six carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl; isobutyl, and n-pentyl; alkyl carbonates having from one to sixcarbon atoms and substituted with one or more halogen atoms such as2,2,2-trichloroethoxymethyl and 2,2,2-trichloro-ethyl; alkenylcarbonates having from two to six carbon atoms such as vinyl and allyl;cycloalkyl carbonates having from three to six carbon atoms such ascyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and phenyl orbenzyl carbonates optionally substituted on the ring with one or moreC₁₋₆ alkoxy, or nitro.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a plant derivednaphthoquinone, generally extracted from the roots of Plumbago speciesof three major phylogenetic families viz. Plumbaginaceae, Droseraceae,and Ebenceae, exhibits highly potent biological activities. The compoundis well known for its general anti-cancer activity. See, for example,Kuo P L, et al. Mol Cancer Ther 2006, 5:3209-3221; Aziz M H, et al.Cancer Res 2008, 68:9024-9032; Shih Y W, et al. Hepatol Res 2009,39:998-1009; Srinivas P, et al. Mol Carcinog 2004, 40:201-211; Powolny AA and Singh S V Pharm Res 2008, 25:2171-2180, each of which isincorporated by reference in its entirety. Since its first reportedapoptotic activities, the compound has been envisaged as a “lead”molecule for the development of new therapeutic agents for cancer.Efforts have focused on the design and synthesis of novel analogues andderivatives of plumbagin which can exhibit better activity, reducedtoxicity, or improved pharmokinetics.

The derivatives of plumbagin described here have been evaluated forapoptotic properties and have been shown to have unexpected selectivitycompared to plumbagin itself. Examples of derivatives of plumbagin thathave been synthesized include those prepared by Mathew et al. byfollowing the general esterification methods, which were previouslystudied for their anti-tuberculosis activity. See, for example, MathewR, et al. Chem Biol Drug Des 2010, 76:34-42, which is incorporated byreference in its entirety.

Derivatives of plumbagin can be tested for anti-cancer potential.Apoptotic potential of the derivatives and plumbagin are evaluated infive human cancer cell lines, such as HepG2 (liver carcinoma), HeLa(cervical carcinoma), MCF-7 (ER-positive) (breast carcinoma), BT-20(ER-negative) (breast carcinoma) or DU145 (prostrate carcinoma), alongwith BJ (normal skin fibroblasts) in vitro using MTT and APOPercentageassays. Certain plumbagin derivatives showed significant selectivecytotoxicity against cancer cell lines although normal cells (BJ) areunaffected even at higher concentration.

The derivatives of plumbagin can be cytotoxic to human breast cancercells. By comparison to normal human cells, the compounds can be 2, 3,4, 5, 8, 10, 15, 20 or more times less cytotoxic to normal human cellscompared to human breast cancer cells. In certain embodiments, the IC₅₀value of the disclosed compounds can be less than 20 micromolar, lessthan 15 micromolar, less than 10 micromolar, or less than 8 micromolar.

A derivative of plumbagin, or a pharmaceutically acceptable saltthereof, can be represented by formula (I) or formula (II):

R¹ can be H, substituted or unsubstituted C₁-C₁₂alkyl, cyano, halo,carboxyl, or nitro.

R² can be H, substituted or unsubstituted C₁-C₁₂alkyl, or O—R′.

R³ can be H, substituted or unsubstituted C₁-C₁₂alkyl, —N(R′)₂, or—O—R′.

R⁴ can be H, —O—R′, or C₁-C₆ alkyl.

Each R′, when present, independently, can be H, substituted orunsubstituted C₁-C₆alkyl, or a hydrolyzable moiety, such as acyl ortrialkylsilyloxy.

In certain embodiments of the compound of formula (I), the double bondbetween R³ and R⁴ can be replaced with a single oxygen (to form an epoxygroup) or H and CN, respectively.

In certain embodiments, R¹ is H.

In certain embodiments, R² is R^(a)—C(O)—O—, in which R^(a) is methyl,ethyl, propyl, propenyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl,hexyl, or phenyl.

In certain embodiments, R³ is H.

In certain embodiments, R⁴ can be H, —O—R′ or C₁-C₆ alkyl.

In certain embodiments, R¹ is H and R³ is H.

In certain embodiments, R² is R^(a)—C(O)—O—, in which R^(a) is methyl,ethyl, propyl, propenyl, isopropyl, butyl, aryl or heteroaryl, forexample, phenyl.

In certain embodiments, R⁴ is H, OH, or methyl.

Each group R¹-R⁵, for each occurrence, can be, independently, optionallysubstituted with halo, carboxylic acid, cyano, or nitro.

In other embodiments, the compound, or a pharmaceutically acceptablesalt thereof, can be represented by one of the following formulae:

The hydroquinonoid, nitro, cyano, and methyl ester derivatives ofplumbagin have been studied for their anti-tumor and anti-leishmanialactivities. See, for example, Phytother Res 2002; 16:133-137, which isincorporated by reference in its entirety. These hydroquinonoid, nitro,cyano, and methyl ester derivatives of compounds can have unexpectedbenefits in treating certain cancers.

In another example, an amino acid moiety derivatives of plumbagin(formula (III)) have been synthesized and subsequently screened forantifeedant activity in tobacco caterpillar (Spodoptera litura) andcastor semi-looper (Achaea janata). See, for example, J Agric Food Chem2009; 57:6090-6094, which is incorporated by reference in its entirety.These amino acid moiety derivatives of plumbagin can have unexpectedbenefits in treating certain cancers.

In another example, a derivative compound can be a naphthoquinonederivatives of plumbagin, which were mostly substituted at C-3 positionthrough carbon-carbon bond formation, synthesized and screened for theirichthyotoxicity. See, for example, Chem Pharm Bull 1997; 45:437-445,which is incorporated by reference in its entirety. These naphthoquinonederivatives of plumbagin can have unexpected benefits in treatingcertain cancers. See, for example, formula (IV), formula (V) or formula(VI).

In some embodiments, the derivative of plumbagin can be a plumbaginhomologue (2-alkyl-1,4-naphthoquinones), including a 3-methylderivative, which has also been synthesized to evaluate theirprostaglandin synthetase (PGS)-inhibition activity. See, for example,Arzneimittelforschung 1984; 34:652-658, which is incorporated byreference in its entirety. These plumbagin homologues can haveunexpected benefits in treating certain cancers. See, for example,formula (VII), formula (VII), formula (IX), formula (X), or formula(XI).

A salt of any of the compounds can be prepared. For example, apharmaceutically acceptable salt can be formed when an amino-containingcompound of this invention reacts with an inorganic or organic acid.Some examples of such an acid include hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Examples of pharmaceutically acceptablesalts thus formed include sulfate, pyrosulfate bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, α-glycerophosphate, sulfate, nitrate, bicarbonate, orcarbonate salts. A compound described herein may also form apharmaceutically acceptable salt when a compound having an acid moietyreacts with an inorganic or organic base. Such salts include thosederived from inorganic or organic bases, e.g., alkali metal salts suchas sodium, potassium, or lithium salts; alkaline earth metal salts suchas calcium or magnesium salts; or ammonium salts or salts of organicbases such as morpholine, piperidine, pyridine, dimethylamine, ordiethylamine salts.

It should be recognized that a suitable compound can contain chiralcarbon atoms. In other words, it may have optical isomers (enantiomers)or diastereoisomers.

The term “cancer” is used throughout the specification to refer to thepathological process that results in the formation and growth of acancerous or malignant neoplasm, i.e., abnormal tissue that grows bycellular proliferation, often more rapidly than normal and continues togrow after the stimuli that initiated the new growth cease. Cancersgenerally show partial or complete lack of structural organization andfunctional coordination with the normal tissue and most invadesurrounding tissues, metastasize to several sites, and are likely torecur after attempted removal and to cause the death of the patientunless adequately treated. As used herein, the term cancer is used todescribe all cancerous disease states applicable to treatment accordingto the present invention and embraces or encompasses the pathologicalprocess associated with all virtually all epithelial cancers, includingcarcinomas, malignant hematogenous, ascitic and solid tumors. Examplesof cancers which may be treated using methods according to the presentinvention include, without limitation, carcinomas (e.g., squamous-cellcarcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cellcarcinomas), particularly those of the bladder, bowel, breast, cervix,colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas,prostate, and stomach; leukemias; benign and malignant lymphomas,particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign andmalignant melanomas; myeloproliferative diseases; sarcomas, particularlyEwing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumorsof the central nervous system (e.g., gliomas, astrocytomas,oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas,ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors,meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas);germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer,cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicularcancer, thyroid cancer, astrocytoma, esophageal cancer, pancreaticcancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixedtypes of neoplasias, particularly carcinosarcoma and Hodgkin's disease;and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas.See, for example, The Merck Manual of Diagnosis and Therapy, 17.sup.thed. (Whitehouse Station, N.J.: Merck Research Laboratories, 1999)973-74, 976, 986, 988, 991).

In addition to the treatment of ectopic cancers as described above, thepresent invention also may be used preferably to treat eutopic cancerssuch as choriocarcinoma, testicular choriocarcinoma, non-seminomatousgerm cell testicular cancer, placental cancer (trophoblastic tumor) andembryonal cancer, among others.

The term “additional anticancer agent” includes chemotherapeutic agentsselected from the group consisting of microtubule-stabilizing agents,microtubule-disruptor agents, alkylating agents, antimetabolites,epidophyllotoxins, antineoplastic enzymes, topoisomerase inhibitors,inhibitors of cell cycle progression, and platinum coordinationcomplexes. These may be selected from the group consisting ofeverolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101 pazopanib,GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107,TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457,MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFRinhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1modulator, a Bc1-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, aPARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TKinhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKTinhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focaladhesion kinase inhibitor, a Map kinase (mek) inhibitor, a VEGF trapantibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402, lucanthone, LY 317615,neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311,romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat,etoposide, gemcitabine, doxorubicin, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258,);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, teniposide, testosterone,thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001 ABT-578,BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin,ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin,granulocyte colony-stimulating factor, zolendronate, prednisone,cetuximab, granulocyte macrophage colony-stimulating factor, histrelin,pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferonalfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, among others.

Formulations containing the compounds according to the present inventionmay take the form of liquid, solid, semi-solid or lyophilized powderforms, such as, for example, solutions, suspensions, emulsions,sustained-release formulations, tablets, capsules, powders,suppositories, creams, ointments, lotions, aerosols, patches or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages.

Pharmaceutical compositions according to the present invention typicallyinclude a conventional pharmaceutical carrier or excipient and mayadditionally include other medicinal agents, carriers, adjuvants,additives and the like. The weight percentage ratio of the one or moreactive ingredients to the one or more excipients can be between about20:1 to about 1:60, or between about 15:1 to about 1:45, or betweenabout 10:1 to about 1:40, or between about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1 or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10,1:15, 1:20, 1:25, 1:30, or 1:35, and preferably is about 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1or 5:1. In some embodiments, formulations of the invention comprisebetween about 250 mg to about 500 mg, or between about 300 mg to about450 mg, or about 325 mg to about 425 mg of total active ingredients andmay optionally contain one or more suitable pharmaceutical excipients.

An injectable composition for parenteral administration (e.g.intravenous, intramuscular or intrathecal) will typically contain thecompound in a suitable i.v. solution, such as sterile physiological saltsolution. The composition may also be formulated as a suspension in anaqueous emulsion.

Liquid compositions can be prepared by dissolving or dispersing thepharmaceutical composition comprising e.g., a CTEP inhibitor, andoptional pharmaceutical adjuvants, in a carrier, such as, for example,aqueous saline, aqueous dextrose, glycerol, or ethanol, to form asolution or suspension. For use in an oral liquid preparation, thecomposition may be prepared as a solution, suspension, emulsion, orsyrup, being supplied either in liquid form or a dried form suitable forhydration in water or normal saline.

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. If desired, the composition may also contain minor amounts ofnon-toxic auxiliary substances such as wetting agents, emulsifyingagents, or buffers.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

Methods for preparing such dosage forms are known or are apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (17th Ed., Mack Pub. Co. 1985). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically effective amount for therapeutic use in a biologicalsystem, including a patient or subject according to the presentinvention.

Methods of treating patients or subjects in need for a particulardisease state or infection comprise administration of an effectiveamount of a pharmaceutical composition comprising therapeutic amounts ofone or more of the novel compounds described herein and optionally atleast one additional bioactive (e.g. anti-cancer) agent according to thepresent invention. The amount of active ingredient(s) (including e.g., aCTEP inhibitor) used in the methods of treatment of the instantinvention that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated, theparticular mode of administration. For example, the compositions couldbe formulated so that a therapeutically effective dosage of betweenabout 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90 or 100 mg/kg of patient/day or in some embodiments,greater than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200mg/kg of the novel compounds can be administered to a patient receivingthese compositions.

Preferably, pharmaceutical compositions in dosage form according to thepresent invention comprise a therapeutically effective amount of atleast 25 mg of e.g., a CTEP inhibitor, at least 50 mg of e.g., a CTEPinhibitor, at least 60 mg of e.g., a CTEP inhibitor, at least 75 mg ofe.g., a CTEP inhibitor, at least 100 mg of e.g., a CTEP inhibitor, atleast 150 mg of e.g., a CTEP inhibitor, at least 200 mg of e.g., a CTEPinhibitor, at least 250 mg of e.g., a CTEP inhibitor, at least 300 mg ofe.g., a CTEP inhibitor, about 350 mg of e.g., a CTEP inhibitor, about400 mg of e.g., a CTEP inhibitor, about 500 mg of e.g., a CTEPinhibitor, about 750 mg of e.g., a CTEP inhibitor, about 1 g (1,000 mg)of e.g., a CTEP inhibitor, alone or in combination with atherapeutically effective amount of at least one additional anti-canceragent.

Preferred embodiments of the pharmaceutical compositions of theinvention comprise between about 250 mg to about 500 mg, or betweenabout 300 mg to about 450 mg, or about 325 mg to about 425 mg, mostpreferably about 380 mg of e.g., a CTEP inhibitor.

The dose of a derivative of plumbagin administered to a subject can beless than 10 μg, less than 25 μg, less than 50 μg, less than 75 μg, lessthan 0.10 mg, less than 0.25 mg, less than 0.5 mg, less than 1 mg, lessthan 2.5 mg, less than 5 mg, less than 10 mg, less than 15 mg, less than20 mg, less than 50 mg, less than 75 mg, less than 100 mg, or less than500 mg.

The activities of a compound described herein can be evaluated bymethods known in the art, e.g., MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,APOPercentage, clonogenic assay, ATP assay, or Extreme Drug Resistance(EDR) assay. See Freuhauf, J. P. and Manetta, A., ChemosensitivityTesting in Gynecologic Malignancies and Breast Cancer 19, 39-52 (1994),which is incorporated by reference in its entirety. The results are thenplotted to generate drug response curves, which allow IC₅₀ values (theconcentration of a compound required to inhibit 50% of the population ofthe treated cells) to be determined. The amount of the compound, or anactive salt or derivative thereof, required for use in treatment canvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and can be ultimately at the discretion ofthe attendant physician or clinician. In general, however, a dose can bein the range of from about 0.01 to about 10 mg/kg of body weight perday.

Other anti-cancer assays are well-known in the art, including in vitroexposure of agents to tumor cells and in vivo antitumor assays in rodentmodels and rarely, in larger animals.

Purely by way of example, comparing measured cancer cell sampleviability, MOMP levels and/or levels of apoptosis-associated proteinswith cell viability, MOMP levels and/or levels of apoptosis-associatedproteins in a control cancer cell sample and comparing measured cancercell CETP levels with CTEP levels of a control cancer cell sample caninclude comparative level differences of about between about 5-10%, orabout 10-15%, or about 15-20%, or about 20-25%, or about 25-30%, orabout 30-35%, or about 35-40%, or about 40-45%, or about 45-50%, orabout 50-55%, or about 55-60%, or about 60-65%, or about 65-70%, orabout 70-75%, or about 75-80%, or about 80-85%, or about 85-90%, orabout 90-95%, or about 95-100%, or about 100-110%, or about 110-120%, orabout 120-130%, or about 130-140%, or about 140-150%, or about 150-160%,or about 160-170%, or about 170-180%, or about 180-190%, or 190-200%, or200-210%, or 210-220%, or 220-230%, or 230-240%, or 240-250%, or250-260%, or about 260-270%, or about 270-280%, or about 280-290%, orabout 290-300%, or differences of about between about +50% to about±0.5%, or about ±45% to about ±1%, or about ±40% to about 1.5%, or about±35% to about ±2.0%, or about ±30% to about ±2.5%, or about ±25% toabout ±3.0%, or about ±20% to about ±3.5%, or about ±15% to about +4.0%,or about ±10% to about +5.0%, or about ±9% to about ±1.0%, or about ±8%to about ±2%, or about ±7% to about ±3%, or about ±6% to about +5%, orabout ±5%, or about ±4.5%, or about ±4.0%, or about ±3.5%, or about±3.0%, or about +2.5%, or about ±2.0%, or about +1.5%, or about ±1.0%.

A “biomarker” is any gene or protein whose level of expression in abiological sample is altered compared to that of a pre-determined level.The pre-determined level can be a level found in a biological samplefrom a normal or healthy subject. Biomarkers include genes and proteins,and variants and fragments thereof. Such biomarkers include DNAcomprising the entire or partial sequence of the nucleic acid sequenceencoding the biomarker, or the complement of such a sequence. Thebiomarker nucleic acids also include RNA comprising the entire orpartial sequence of any of the nucleic acid sequences of interest. Abiomarker protein is a protein encoded by or corresponding to a DNAbiomarker of the invention. A biomarker protein comprises the entire orpartial amino acid sequence of any of the biomarker proteins orpolypeptides. Biomarkers can be detected, e.g. by nucleic acidhybridization, antibody binding, activity assays, polymerase chainreaction (PCR), Si nuclease assay and gene chip.

A “control” as used herein may be a positive or negative control asknown in the art and can refer to a control cell, tissue, sample, orsubject. The control may, for example, be examined at precisely ornearly the same time the test cell, tissue, sample, or subject isexamined. The control may also, for example, be examined at a timedistant from the time at which the test cell, tissue, sample, or subjectis examined, and the results of the examination of the control may berecorded so that the recorded results may be compared with resultsobtained by examination of a test cell, tissue, sample, or subject. Forinstance, as can be appreciated by a skilled artisan, a control maycomprise data from one or more control subjects that is stored in areference database. The control may be a subject who is similar to thetest subject (for instance, may be of the same gender, same race, samegeneral age and/or same general health) but who is known to not have afibrotic disease. As can be appreciated by a skilled artisan, themethods of the invention can also be modified to compare a test subjectto a control subject who is similar to the test subject (for instance,may be of the same gender, same race, same general age and/or samegeneral health) but who is known to express symptoms of a disease. Inthis embodiment, a diagnosis of a disease or staging of a disease can bemade by determining whether protein or gene expression levels asdescribed herein are statistically similar between the test and controlsubjects.

The terms “level” and/or “activity” as used herein further refer to geneand protein expression levels or gene or protein activity. For example,gene expression can be defined as the utilization of the informationcontained in a gene by transcription and translation leading to theproduction of a gene product.

In certain non-limiting embodiments, an increase or a decrease in asubject or test sample of the level of measured biomarkers (e.g.proteins or gene expression) as compared to a comparable level ofmeasured proteins or gene expression in a control subject or sample canbe an increase or decrease in the magnitude of approximately5,000-10,000%, or approximately ±2,500-5,000%, or approximately+1,000-2,500%, or approximately ±500-1,000%, or approximately +250-500%,or approximately ±100-250%, or approximately 50-100%, or approximately+25-50%, or approximately ±10-25%, or approximately ±10-20%, orapproximately ±10-15%, or approximately +5-10%, or approximately ±1-5%,or approximately ±0.5-1%, or approximately +0.1-0.5%, or approximately±0.01-0.1%, or approximately ±0.001-0.01%, or approximately±0.0001-0.001%.

The values obtained from controls are reference values representing aknown health status and the values obtained from test samples orsubjects are reference values representing a known disease status. Theterm “control”, as used herein, can mean a sample of preferably the samesource (e.g. blood, serum, tissue etc.) which is obtained from at leastone healthy subject to be compared to the sample to be analyzed. Inorder to receive comparable results the control as well as the sampleshould be obtained, handled and treated in the same way. In certainexamples, the number of healthy individuals used to obtain a controlvalue may be at least one, preferably at least two, more preferably atleast five, most preferably at least ten, in particular at least twenty.However, the values may also be obtained from at least one hundred, onethousand or ten thousand individuals.

A level and/or an activity and/or expression of a translation product ofa gene and/or of a fragment, or derivative, or variant of saidtranslation product, and/or the level or activity of said translationproduct, and/or of a fragment, or derivative, or variant thereof, can bedetected using an immunoassay, an activity assay, and/or a bindingassay. These assays can measure the amount of binding between saidprotein molecule and an anti-protein antibody by the use of enzymatic,chromodynamic, radioactive, magnetic, or luminescent labels which areattached to either the anti-protein antibody or a secondary antibodywhich binds the anti-protein antibody. In addition, other high affinityligands may be used. Immunoassays which can be used include e.g. ELISAs,Western blots and other techniques known to those of ordinary skill inthe art (see Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 andEdwards R, Immunodiagnostics: A Practical Approach, Oxford UniversityPress, Oxford; England, 1999). All these detection techniques may alsobe employed in the format of microarrays, protein-arrays, antibodymicroarrays, tissue microarrays, electronic biochip or protein-chipbased technologies (see Schena M., Microarray Biochip Technology, EatonPublishing, Natick, Mass., 2000).

Certain diagnostic and screening methods of the present inventionutilize an antibody, preferably, a monocolonal antibody, capable ofspecifically binding to a protein as described herein or activefragments thereof. The method of utilizing an antibody to measure thelevels of protein allows for non-invasive diagnosis of the pathologicalstates of kidney diseases. In a preferred embodiment of the presentinvention, the antibody is human or is humanized. The preferredantibodies may be used, for example, in standard radioimmunoassays orenzyme-linked immunosorbent assays or other assays which utilizeantibodies for measurement of levels of protein in sample. In aparticular embodiment, the antibodies of the present invention are usedto detect and to measure the levels of protein present in a sample.

Humanized antibodies are antibodies, or antibody fragments, that havethe same binding specificity as a parent antibody, (i.e., typically ofmouse origin) and increased human characteristics. Humanized antibodiesmay be obtained, for example, by chain shuffling or by using phagedisplay technology. For example, a polypeptide comprising a heavy orlight chain variable domain of a non-human antibody specific for adisease related protein is combined with a repertoire of humancomplementary (light or heavy) chain variable domains. Hybrid pairingsspecific for the antigen of interest are selected. Human chains from theselected pairings may then be combined with a repertoire of humancomplementary variable domains (heavy or light) and humanized antibodypolypeptide dimers can be selected for binding specificity for anantigen. Techniques described for generation of humanized antibodiesthat can be used in the method of the present invention are disclosedin, for example, U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761; and5,693,762. Furthermore, techniques described for the production of humanantibodies in transgenic mice are described in, for example, U.S. Pat.Nos. 5,545,806 and 5,569,825.

In order to identify small molecules and other agents useful in thepresent methods for treating a cancer by modulating the activity andexpression of a disease-related protein and biologically activefragments thereof can be used for screening therapeutic compounds in anyof a variety of screening techniques. Fragments employed in suchscreening tests may be free in solution, affixed to a solid support,borne on a cell surface, or located intracellularly. The blocking orreduction of biological activity or the formation of binding complexesbetween the disease-related protein and the agent being tested can bemeasured by methods available in the art.

Other techniques for drug screening which provide for a high throughputscreening of compounds having suitable binding affinity to a protein, orto another target polypeptide useful in modulating, regulating, orinhibiting the expression and/or activity of a disease, are known in theart. For example, microarrays carrying test compounds can be prepared,used, and analyzed using methods available in the art. See, e.g.,Shalon, D. et al., 1995, International Publication No. WO95/35505,Baldeschweiler et al., 1995, International Publication No. WO95/251116;Brennan et al., 1995, U.S. Pat. No. 5,474,796; Heller et al., 1997, U.S.Pat. No. 5,605,662.

Identifying small molecules that modulate protein activity can also beconducted by various other screening techniques, which can also serve toidentify antibodies and other compounds that interact with proteinsidentified herein and can be used as drugs and therapeutics in thepresent methods. See, e.g., Enna et al., eds., 1998, Current Protocolsin Pharmacology, John Wiley & Sons, Inc., New York N.Y. Assays willtypically provide for detectable signals associated with the binding ofthe compound to a protein or cellular target. Binding can be detectedby, for example, fluorophores, enzyme conjugates, and other detectablelabels well known in the art. The results may be qualitative orquantitative.

For screening the compounds for specific binding, various immunoassaysmay be employed for detecting, for example, human or primate antibodiesbound to the cells. Thus, one may use labeled anti-hIg, e.g., anti-hIgM,hIgG or combinations thereof to detect specifically bound humanantibody. Various labels can be used such as radioisotopes, enzymes,fluorescers, chemiluminescers, particles, etc. There are numerouscommercially available kits providing labeled anti-hIg, which may beemployed in accordance with the manufacturer's protocol.

In one embodiment, a kit can comprise: (a) at least one reagent which isselected from the group consisting of (i) reagents that detect atranscription product of the gene coding for a protein marker asdescribed herein (ii) reagents that detect a translation product of thegene coding for proteins, and/or reagents that detect a fragment orderivative or variant of said transcription or translation product; (b)optionally, one or more types of cells, including engineered cells inwhich cellular assays are to be conducted; (c) instructions fordiagnosing, or prognosticating a disease, or determining the propensityor predisposition of a subject to develop such a disease or ofmonitoring the effect of a treatment by determining a level, or anactivity, or both said level and said activity, and/or expression ofsaid transcription product and/or said translation product and/or offragments, derivatives or variants of the foregoing, in a sampleobtained from said subject; and comparing said level and/or saidactivity and/or expression of said transcription product and/or saidtranslation product and/or fragments, derivatives or variants thereof toa reference value representing a known disease status (patient) and/orto a reference value representing a known health status (control) and/orto a reference value; and analyzing whether said level and/or saidactivity and/or expression is varied compared to a reference valuerepresenting a known health status, and/or is similar or equal to areference value representing a known disease status or a referencevalue; and diagnosing or prognosticating a disease, or determining thepropensity or predisposition of said subject to develop such a disease,wherein a varied or altered level, expression or activity, or both saidlevel and said activity, of said transcription product and/or saidtranslation product and/or said fragments, derivatives or variantsthereof compared to a reference value representing a known health status(control) and/or wherein a level, or activity, or both said level andsaid activity, of said transcription product and/or said translationproduct and/or said fragments, derivatives or variants thereof issimilar or equal to a reference value and/or to a reference valuerepresenting a known disease stage, indicates a diagnosis or prognosisof a disease, or an increased propensity or predisposition of developingsuch a disease, a high risk of developing signs and symptoms of adisease.

Reagents that selectively detect a transcription product and/or atranslation product of the gene coding for proteins can be sequences ofvarious length, fragments of sequences, antibodies, aptamers, siRNA,microRNA, and ribozymes. Such reagents may be used also to detectfragments, derivatives or variants thereof.

In one embodiment, the invention provides a method of treating a subjectwho suffers from a cancer, the method comprising administering to thesubject a therapeutically effective amount of Cholesteryl Ester TransferProtein (CETP) inhibitor selected from the group consisting of acetylplumbagin, rosuvastatin, rivastatin, pitavastatin, lovastatin,simvastatin, pravastatin, fluvastatin, dalceptrpib, anacetrapib,evacetrapib, torcetrapib, atorvastatin (preferably atorvastatinhemi-calcium), cerivastatin, CETP inhibitors described in U.S. Pat. Nos.7,652,049, 6,140,343, 6,197,786, 6,723,752 (preferably(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol),and U.S. Pat. No. 5,512,548, CETP-inhibitory rosenonolactone derivativesand phosphate-containing analogs of cholesteryl ester described in J.Antibiot., 49(8): 815-816 (1996) and Bioorg. Med. Chem. Lett.;6:1951-1954 (1996), propanethioic acid, 2-methyl-, S-[2-[[[1-(2ethylbutyl)cyclohexyl]carbonyl]amino]phenyl]ester (Dalcetrapib),S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-3-phenylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]3-pyridinethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]chlorothioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]methoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]phenoxy-thioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclopropanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-4-carbamoylthiobutyrate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-hydroxy-2-methylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]thioacetate;S-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S[4,5-dichloro-2-(1-isopentylcyclopentanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;O-methyl S-[2-(1-isopentylcyclohexanecarbonylaminophenylmonothiocarbonate;S-[2-(1-methylcyclohexanecarbonylamino)phenyl]S-phenyldithiocarbonate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]N-phenylthiocarbarnate;S-[2-(pivaloylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(2-cyclohexylpropionylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-pentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclohexylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopropylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcycloheptanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcyclobutanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-nitrophenyl]2,2-dimethylthiopropionate;S-[4-cyano-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[5-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-difluoro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[5-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;bis-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]disulfide;2-tetrahydrofurylmethyl-2-(1-isopentylcyclohexanecarbonylamino)phenyldisulfide; N-(2-mercaptophenyl)-1-ethylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-propylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-butylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-isobutylcyclohexanecarboxamide;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclohexanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiobenzoate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]5-carboxythiopentanoate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-methylphenyl]thioacetate;bis-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]disulfide;N-(2-mercaptophenyl)-1-(2-ethylbutyl)cyclohexanecarboxamide;S[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-methylthiopropionate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate-;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]-acetylpiperidine-4-thiocarboxylate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]thioacetate;S-[2-[1(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2,2-dimethylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]methoxythio-acetate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-hydroxy-2-methylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]4-chlorophenoxythioacetate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;andS-[2-(1-isobutylcyclohexanecarbonylarnino)phenyl]-1-acetyl-piperidine-4-thiocarboxylateand analogs, derivatives, pharmaceutically acceptable salts,enantiomers, diastereomers, solvates and polymorphs thereof.

Preferred Cholesteryl Ester Transfer Protein (CETP) inhibitors includecompounds selected from the group consisting of rosuvastatin,rivastatin, pitavastatin, lovastatin, simvastatin, pravastatin,fluvastatin, dalceptrpib, anacetrapib, evacetrapib, torcetrapib,atorvastatin (preferably atorvastatin hemi-calcium), cerivastatin andanalogs, derivatives, pharmaceutically acceptable salts, enantiomers,diastereomers, solvates and polymorphs thereof.

In certain embodiments, a subject suffering from a cancer isadministered or co-administered one or more anticancer agents selectedfrom the group consisting of tamoxifen, paclitaxel, fluorouracil (5-FU),plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), or an analog,derivative, pharmaceutically acceptable salt, enantiomer, diastereomer,solvate or polymorph thereof. The subject may suffer from a form ofrefractory breast cancer, may have developed an acquired anti-estrogenresistance and/or may exhibit an intrinsic resistance to anti-estrogenand anti-HER2 therapies.

In certain embodiments, a subject is co-administered a therapeuticallyeffective amount of:

(a) acetyl plumbagin;(b) one or more additional anticancer agents selected from the groupconsisting of tamoxifen, paclitaxel and fluorouracil (5-FU); and(c) optionally, a Cholesteryl Ester Transfer Protein (CETP) inhibitorselected from the group consisting of rosuvastatin, rivastatin,pitavastatin, lovastatin, simvastatin, pravastatin, fluvastatin,dalceptrpib, anacetrapib, evacetrapib, torcetrapib, atorvastatin(preferably atorvastatin hemi-calcium), cerivastatin and analogs,derivatives, pharmaceutically acceptable salts, enantiomers,diastereomers, solvates and polymorphs thereof.

Other embodiments provide a method of improving the clinical outcome ofa subject who suffers from a cancer and who is undergoing treatment withone or more anticancer agents selected from the group consisting oftamoxifen, paclitaxel and fluorouracil (5-FU), the method comprisingco-administering to the subject one or more compounds selected from thegroup consisting of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), acompound of formula (I) or formula (II), and the analogs, derivatives,pharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof:

wherein R′ is H, substituted or unsubstituted C₁-C₁₂alkyl, cyano, halo,carboxyl, or nitro;

R² is H, substituted or unsubstituted C₁-C₁₂alkyl, or O—R′;

R³ is H, substituted or unsubstituted C₁-C₁₂alkyl, —N(R′)₂, or —O—R′;

R⁴ is H, —O—R′, or C₁-C₆ alkyl; and

each R′, when present, independently, is H, substituted or unsubstitutedC₁-C₆alkyl, or a hydrolyzable moiety, such as acyl or trialkylsilyloxy.

In still other embodiments, a subject who suffers from a cancer isadministered a therapeutically effective amount of one or morecompositions selected from the group consisting of a Cholesteryl EsterTransfer Protein (CETP) inhibiting antisense oligonucleotide, aCholesteryl Ester Transfer Protein (CETP) inhibiting siRNA and ananti-Cholesteryl Ester Transfer Protein (CETP) antibody.

One illustrative Cholesteryl Ester Transfer Protein (CETP) inhibitingantisense oligonucleotide which can be used in methods and compositionsof the invention has the sequence (5′-CAGCACTTTAATGCCAGTGG-3′), whereinthe sequence contains 2′-O-methoxyethyl (2′ MOE) groups at positions 1-5and 15-20 which are targeted to human CETP. See Bell, et al., October2013, The Journal of Lipid Research, 54, 2647-2657. Other illustrativeCholesteryl Ester Transfer Protein (CETP) inhibiting antisenseoligonucleotide have one of the sequences identified as SEQ ID NOS. 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 and 50 of PCT WO2003014306, wherein such sequences optionallycomprises at least one modified internucleoside linkage (e.g. aphosphorothioate linkage) and/or at least one modified sugar moiety(e.g. a 2′-O-methoxyethyl sugar moiety), and/or at least one modifiednucleobase (e.g. a 5-methylcytosine). In some embodiments, theoligonucleotide has at least about 70%, 75%, 80%, 85%, 90% or 95%complementarity with a nucleic acid encoding human Cholesteryl EsterTransfer Protein (CETP). In other illustrative embodiments, theoligonucleotide specifically hybridizes with a nucleotide sequenceencoding human Cholesteryl Ester Transfer Protein (CETP), wherein thenucleotide sequence comprises a translation initiation codon, atermination codon, a coding region, a 5′ untranslated region, a 3′untranslated region, an intron:exon junction or an exon:intron junction.

In other embodiments, a subject suffering from a cancer is administereda Cholesteryl Ester Transfer Protein (CETP) inhibiting double strandedshort interfering RNA (ds siRNA), wherein one strand of thedouble-stranded siRNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a CETP gene or a portionthereof, and wherein a second strand of the double-stranded siRNAmolecule comprises a nucleotide sequence that is complementary to anucleotide sequence of a CETP gene RNA or a portion thereof. Anillustrative Cholesteryl Ester Transfer Protein (CETP) inhibiting doublestranded short interfering RNA (ds siRNA) can have at least about 70%,75%, 80%, 85%, 90% or 95% complementarity with a CTEP inhibiting dssiRNA described in United States Patent Application Document No.20050171040, the complete disclosure of which is hereby incorporated byreference.

In still other embodiments, a subject who suffers from a cancer isadministered a Cholesteryl Ester Transfer Protein (CETP) inhibitingantibody or a fragment thereof.

Preferred Cholesteryl Ester Transfer Protein (CETP) inhibitingantibodies include a humanized monoclonal antibody or a F(ab′)₂ or Fab′fragment thereof, and the CTEP inhibiting antibodies and antibodyfragments described or referenced in United States Patent ApplicationDocument No. 20140328851, the complete disclosure of which is herebyincorporated by reference.

The invention is illustrated further in the following non-limitingexamples.

Example 1 PL and AP Induce Intrinsic Apoptosis in MCF-7 Cells

We have previously shown that Acetyl Plumbagin (AP) has selectiveactivity against MCF-7 (ER positive breast cancer (BC) cells) ascompared to normal skin fibroblasts (BJ) and triple negative BT20 BCcells [1]. We further sought to characterize the timeline of molecularevents and specific pathway of apoptosis induced by AP in comparison toits parent molecule PL in MCF-7 cells. As a first step, MCF-7 cells weretreated with 1, 2.5, 5 and 10 μM PL or AP for 24 h and percentageviability was determined after 24 h using the MTT assay. Both 1 and 2.5μM concentrations of PL and AP had no significant effect on cellviability however 5 and 10 μM reduced cell viability by 80% or greater(FIG. 1A). Next the effect of 10 μM PL drug on cell viability over timewas determined. PL and AP reduced cell viability to 50% in as early as 6h (FIG. 1B). The percentage of cells undergoing apoptosis at the earlytime points i.e. 2 and 6 h was determined by the APOPercentage assay.APOPercentage dye binds to extracellularly exposed phosphatidylserine(PS) on the plasma membrane of apoptotic cells. After induction with PLand AP, an approximately similar level of apoptosis i.e. 20% and 45% wasobserved at 2 and 6 h respectively (FIG. 1C).

Since 50% of cells were observed to undergo apoptosis within 6 h oftreatment, further studies were performed within this timeframe toestablish the chronology of apoptotic events. The changes inMitochondrial Outer Membrane Potential (MOMP) and proteins involved inthe apoptotic cascade were assessed (FIG. 2). Untreated cells yieldedhigher fluorescence in the FL-2 channel while lower fluorescent values,typical of disrupted MOMP, were observed in cells (FIG. 2A). treatedwith 10 μM PL, AP and 100 mM H₂O₂ (as a positive control).

Intrinsic apoptosis is activated by the mitochondria where MOMP isreduced and the imbalance between pro- and anti-apoptotic factorsresults in activation of caspases 9, 3 and 7, cleavage of caspasesubstrate like PARP-1 (Poly [ADP-ribose] polymerase 1) and fragmentationof DNA. After treatment with 10 μM PL and AP alterations in the ratio ofBax and Bcl2 were observed and pro-caspase 9 levels were reduced (FIGS.2B and C). A reduction of pro-caspase 7 was observed as early as 2 and 6h in cells treated with PL while pro-caspase 7 was reduced at 12 h incells treated with AP (FIGS. 2D and E). cl-PARP-1 a substrate of caspase3/7 was substantially reduced from full length to the cleaved fragmentat the 12 and 24 h time points. The expression of the DNA damage markerpH2AX was significantly increased as early as 2 h in both PL and APtreated MCF-7 cells and remained elevated for the duration of theexperiment. The data suggests that both PL and AP activate the intrinsicapoptosis pathway in MCF-7 cells.

Example 2 In Vivo Efficacy and Toxicity Profiles in Mouse MCF-7Xenograft Model of BC

Given that PL and AP activate the intrinsic apoptotic pathway and thatAP displayed selectivity for ER positive BC with low cytotoxicity tonormal cells in vitro [1], we asked whether AP has similar effects invivo. MCF-7 tumors grown up to 300 mm3 in four nude mice were harvested,dissected into 1-2 mm3 fragments and implanted into the study group forfurther testing. Nude mice (with tumor volume of 150 mm3) were treatedwith 25% Polyethylene glycol (PEG) 400 as vehicle, 2 mg/kg PL and 5mg/kg AP for 21 days during which tumor volume, body weight and, at thetime of sacrifice, tumor weight was measured. Initially mice weretreated with 5 mg/kg PL however within four to six days of treatment 8mice succumbed to treatment due to toxicity hence the well-tolerated PLdose of 2 mg/kg was used (data not shown). Although 2 mg/kg PL reducedtumor volume by approximately 17%, no difference was observed for tumorweight in comparison to vehicle (FIG. 3A). Treatment with 5 mg/kg AP for21 days inhibited tumor growth by 45% and reduced tumor volume by 37%and in nude mice (FIGS. 3A and B). None of the animals of vehicle groupexhibited tumor regression. All 5 animals in vehicle and AP treatedgroup survived whereas one mouse died in PL treated group even at 2mg/kg dose. The body weight of mice treated with vehicle and AP remainedunaffected by treatment however mice treated with PL loss 2.5 g (12%) ofbody weight within the first 3 days which recovered slightly andremained stable with time (FIG. 3C). When liver tissue is damaged orinflamed liver cells tend to leak enzymes into the blood. Elevatedactivity of Alanine transaminase (ALT) and Aspartate transaminase (AST)liver enzyme markers in serum or blood is indicative of stress andtoxicity. PL treatment increased activity of AST and ALT enzymes in mice(FIGS. 3D and E). On the contrary, AP treatment reduced the ALT and ASTactivity by 36% and 7%, respectively.

Example 3 PL and AP Disrupt Lipid Rafts and Deplete MCF-7 Cells ofCholesterol

Cholesterol plays an integral role in the formation of lipid rafts,steroid hormone synthesis and cell membrane formation [2,3]. Publisheddata suggests a link between cholesterol levels and cancer whereelevated cholesterol levels have been observed before and around thetime of breast cancer diagnosis [4,5,6]. Cancer cells have a greateraffinity for cholesterol to pace with demands of proliferation andsynthesis of new membranes and lipid rafts. Pathway predictions derivedfrom microarray expression data pointed at the impairment of cholesterolrelated processes by AP. To confirm whether cholesterol related pathwayswere affected by PL or AP, we performed lipid raft staining in MCF-7cells and cholesterol quantitation in both MCF-7 cells and plasmasamples obtained from mice. Lipid rafts of MCF-7 cells were stainedusing a conjugate of cholera toxin subunit B (CT-B) which binds to thepentasaccharide chain of ganglioside GM1 present in plasma membrane thatselectively partitions into lipid rafts [7,8].

A distinct membrane staining representing lipid rafts was observed inuntreated cells however in the presence of 10 μM PL or AP over time thedistribution of lipid rafts (GM1 disappearance from plasma membranes)was disrupted (FIG. 4). No distinct membrane lipid raft staining wasobserved in BJ cells as normal cells do not accumulate cholesterol andover express lipid rafts (Data not shown). As a result no adverseeffects were observed on BJ cell morphology when treated with PL or AP.

The redistribution and distortion of lipid rafts suggested that PL andAP interfered with cholesterol associated mechanisms. Next, wedetermined the effects of PL and AP on cholesterol levels in cells.Within 2 h of treatment with PL or AP, total cholesterol was reduced by35% in MCF-7 cells as compared to untreated cells (FIG. 5). To testwhether cholesterol depletion occurred in other cell types or if thiswas limited to ER positive BC cells, BJ and BT20 cells were also treatedwith PL or AP (Fig.). Treatment with 10 μM PL and AP resulted in 3-6%reduction in total cholesterol in BJ and BT20 cells relative to theuntreated cells. The data thus far indicates that both PL and AP actthrough cholesterol modulation and that these effects might be estrogenmediated as total cholesterol in ER negative BJ and BT20 cells remainedunaffected in the presence of PL or AP.

Next we asked if addition of cholesterol can rescue MCF-7 cells fromundergoing apoptosis when treated with PL or AP. The addition ofcholesterol significantly reduced apoptosis in PL (30% to 10%) and AP(20% to negligible) treated cells (FIG. 5B).

Example 4 PL and AP Modulate Cholesterol Levels In Vivo

We next asked whether changes observed in cholesterol levels in vitroare also observable in vivo. Total cholesterol levels in serum of MCF-7xenograft models were measured with the AMPLEX Red kit. Negligiblealterations to total cholesterol were noted for 2 mg/kg PL treatmentafter 21 days when compared to Vehicle control (FIG. 6A). A significantincrease in the amount of cholesterol in serum of mice treated with 5mg/kg AP was observed which suggested cholesterol efflux/depletion fromcells into the serum in the presence of AP in vivo. We then determinedwhether the elevated level of cholesterol noted in mice treated with APwas bound or unbound. Using the AMPLEX Red assay, but excluding thecholesterol esterase enzyme, the level of free cholesterol wasdetermined (FIG. 6B). Although the amount of free cholesterol comparedto the overall total amount of cholesterol in mice was minute, freecholesterol level in AP treated mice was significantly lower than thatof the PL or control groups (FIG. 6B).

The balance between the HDL and LDL ratio has important physiologicalimplications. Excess cholesterol is cleared up from the bloodstream byHDL by reverse cholesterol transport (RCT). HDL also maintains cellularcholesterol homeostasis by unloading excess cholesterol from cells [9].Not surprisingly an imbalance in HDL:VLDL/LDL levels has been correlatedwith the progression of atherosclerosis [10]. A different kit (ABNOVAHDL and VLDL assay kit) and spectrophotomical measurements were employedto determine the levels of total, HDL and LDLNLDL cholesterol. Serumsamples of mice were separated into different fractions as permanufacturer's instructions (FIG. 6C). The ratio of HDL to LDLNLDL inVehicle group was 3.4:1. In the AP treated group total cholesterollevels were also elevated as observed earlier however the ratio of HDLto LDLNLDL (3.1:1) remained similar to that of Vehicle group. On thecontrary PL treated mice, with slightly lower total cholesterol comparedto the Vehicle group, had a ratio of HDL to LDLNLDL of 1.8:1. Our dataso far suggests that although both PL and AP possess cholesterolmodulating effects in vitro and in vivo AP is safer to use as itmaintains the HDL:LDL/VLDL balance.

Example 5 Cholesterol Ester Transfer Protein (CETP): A New Link toCancer

CETP transfers cholesterol esters and triglycerides between plasmalipoproteins and plays an important role in the RCT i.e. transport ofcholesterol to the liver for excretion in order to maintain cholesterolhomeostasis [11]. In the past decade, it was believed that CETPinhibition results in increased HDL levels and reduce the occurrence ofcardiovascular disease (CVD) [12], which led to several clinical trialsto test the anti-atherosclerosis effects of CETP inhibition.Alternatively, it is also reported that CETP also facilitates efflux ofcellular free cholesterol and an increase in plasma CETP activity islinked to an enhanced capacity of plasma to promote cholesterol effluxfrom human macrophages independent of lipid variations [13]. Recently,it has been proposed based on extensive published data that CETPinhibition does not test the increase in HDL hypothesis as hoped, andCETP has a protective effect by accelerating RCT [14]. Thiscontroversial role of CETP calls for more detailed investigations incholesterol related diseases.

Several experimental and clinical studies have also linked thecholesterol [15,16,17], cholesterol metabolites [5,18] and lipoproteins[4,19] to carcinogenesis and tumor development. Since CETP maintainscholesterol homeostasis, we hypothesized that CETP might be fueling thecancer cells by maintaining the cholesterol supply for rapid cell growthand division. Therefore, understanding the role of CETP in cancer isimportant.

We performed qRT-PCR using mRNA isolated from mice tumor samples todetermine the expression of CETP after treatment with PL and AP (FIG.7A). Notably, AP treatment significantly reduced the expression of CETPin tumors by 60%, however, PL had no effect on CETP transcription. CETPmRNA is translated into 74 kD protein comprised of 476 amino acids thatlocalizes in plasma [11]. The nude mice used in this study were CETPnull/deficient hence any CETP levels in serum would reflect thatemanating from the tumors. CETP protein levels in mice serum sampleswere determined by western blotting (FIG. 7B). Densitometry analysis ofCETP protein levels revealed that AP upregulated CETP levels while CETPlevels were slightly reduced in the PL treated group when compared toVehicle (Data not shown).

The activity of CETP in mice plasma was determined using the CETPactivity assay kit according to the manufacturer's instructions (FIG.7D). CETP activity was elevated in AP treated mice compared to Vehicle.This observation was in accordance with the CETP protein expressionlevels observed in Vehicle, AP and PL test groups. The expression levelsof CETP have been reported to be linear with its activity [14].

Example 6 CETP Helps to Maintain Cellular Cholesterol Levels in CancerCells

The in vivo data suggested that AP modulates CETP levels and activity.Since we could only capture the protein expression and activity of CETPin mice serum which did not reflect the effects of CETP ablation intumor cells, we studied the role of CETP in MCF-7 cells in vitro.Transient transfection of MCF-7 cells with siRNA directed to CETPresulted in an 80% or greater inhibition of CETP mRNA (data not shown).We measured the amount of cholesterol in CETP knock-out MCF-7 untreatedand/or treated with 10 μM AP and PL. The total cholesterol was decreasedin untreated CETP siRNA cells by 11%, however AP and PL treatedknock-out cells did not show results any different from control (ctrl)siRNA cells. (FIG. 8). This demonstrates that knocking-out CETP inoptimally growing MCF-7 leads to lowered cholesterol levels in thesecells. No significant changes were observed in cholesterol levels of BJCETP siRNA cells whether treated with PL or AP or left untreated (datanot shown).

Example 7 CETP is Required for Growth of Cancer Cells

We investigated whether CETP is required for optimal growth of cancercells. We silenced the CETP gene in MCF-7 cells and measured theirgrowth over a period of six days. Interestingly, the proliferation ofMCF-7 cells was reduced by 20% (FIG. 9).

Example 8 CETP Acts as an Anti-Apoptotic Gene in Cancer Cells

Since, as shown above, CETP is required for optimal growth of MCF-7cells, we further hypothesized that CETP may help cancer cell toresist/escape apoptotic signals thus preventing cell death. Severalapoptotic markers such as MOMP, phosphatidylserine exposure andcaspase-3/7 activity were tested to establish a link between CETP andapoptosis.

CETP knockdown alone had no observable effects on MMP, however, in thepresence of PL or AP a reduction in MMP of 13 and 17% respectively wasobserved when compared to Ctrl siRNA cells (FIG. 10A). Next, we intendedto see if CETP silencing increased sensitivity of MCF-7 cells toapoptosis. In the presence of PL or AP, CETP knockdown significantlyincreased the caspases-3/7 activity in MCF-7 cells (FIG. 10B). Finally,the level of apoptosis in MCF-7 CETP siRNA cells was found to beincreased by 10% in the presence of PL or AP (FIG. 10C), whereas noincreased sensitivity to apoptosis was observed in BJ CETP knockdowncells treated with PL or AP (FIG. 10D).

Our data suggests that CETP plays a role in cancer cell survival andcholesterol metabolism as knockdown of CETP resulted in an increasedsensitivity to apoptosis and a decrease in total cholesterol. AlthoughCETP is secreted into the extracellular environment andbloodstream/plasma we propose that CETP expression locally plays a rolein cell growth and apoptosis. To get an idea about the mRNA expressionlevels of CETP in breast cancer, we compared the expression of CETP mRNA(as compared to normal) in six published microarray datasets inOncomine. CETP mRNA was over-expressed in these datasets (FIG. 1). Ourresults suggest a link between cholesterol, CETP and breast cancer.

Example 9 Effect of CETP Knockout on Tamoxifen Action in MCF-7 Cells

To test if effect of CETP knockout on apoptosis in MCF-7 is limited toAP, we treated the CETP knockout MCF-7 cells with tamoxifen (a knownfirst line drug for breast cancer treatment). A significant increase incaspase-3/7 activity was observed (FIG. 12). This observation was notlimited to PL and AP as similar observations were made in CETP knockdowncells treated with tamoxifen (FIG. 12).

Example 10 AP Reduce Toxicity and Increase Efficacy of KnownChemotherapeutic Drugs

In our earlier in vivo experiments, we found that AP could even reducethe toxicity linked with vehicle control as demonstrated by ALT and ASTlevels. We anticipated that AP might have the potential to reducetoxicity and increase efficacy of known chemotherapeutic drugs which mayenhance the usage of these drugs at lower concentrations with increasedcancer killing effects. To test this preliminary, we treated normal (BJ)and Cancer (MCF-7) cells with different chemotherapeutic drugs assummarized in FIG. 13.

The results of this experiment show that AP when used in combinationwith 5-Florouracil increases cell viability of normal cells (7%) whileat the same time reduces viability of cancer cells (76%). Similareffects were noticed in the case of paclitaxel. Various concentrationsof Tamoxifen were tested and it was found that AP was unable to rescuenormal cells from toxic effects of tamoxifen at higher concentrationsbut when used with 10 μM of tamoxifen, the viability of normal cells wasincreased by 7% and viability of cancer cells was reduced by 50%. It isalso clear from data that the cancer killing effect achieved using 20 μMtamoxifen was possible to achieve using a combination of AP with 10 μMtamoxifen while enhancing the viability of normal cells at the sametime.

This preliminary data shows that the AP combined with half the dose oftamoxifen can help to achieve long term benefits in terms of reducedtoxicity and enhanced efficacy.

Example 11

CETP: A Target of AP (Evidence that AP Binds to CETP Inside the Cells)

Thermal shift assay (TSA) captures the drug protein binding using thefact that the thermostability of the protein is changed after ligandbinding. We did three runs of TSA [20] to identify direct binding of APwith CETP (FIG. 14). The data shows that in untreated MCF-7 cells, thelevel of protein starts decreasing around 64° C. while CETP seems to bemore stable even at 70° C. in cells treated with 10 μM of AP/PL. Theseresults confirm that AP binds to CETP in the cells. Further studies areneeded to study the effects of thermostability on function of CETP.

CONCLUSIONS

The following can be concluded from the experimental results describedabove:

1. AP has the potential to kill estrogen positive breast cancer cellsboth in vivo and in vitro at dose which is not toxic to normal cells.2. AP kills cancer cells by depleting cholesterol from membranes ofcholesterol enriched cancer cells that triggers cell death pathways.This is confirmed as replenishing cholesterol blocks cell death.3. Depleted cholesterol comes to plasma for clearance. AP maintainsHDL/LDL ratio for optimal clearance of cholesterol.4. AP increases CETP activity (this activity is inversely related tocardiovascular diseases) leading to clearance of excess cholesterol fromplasma to liver.5. Discovered link of CETP to cancer (new cancer biology—never reportedbefore).6. Knocking-out CETP slowed growth of cultured cancer cells by 20%within 6 days.7. CETP knockout also modulated cell death signaling pathways(apoptosis).8. CETP also increased caspases-3/7 activity (these enzymes areexecutioners of apoptotic cell death) linked to Tamoxifen (this drug isa gold standard breast cancer therapy).9. AP binds to CETP inside the MCF-7 cancer cells.10. AP when combined with Tamoxifen, increased the viability of normalcells and significantly decreased the viability of cancer cells. Itmeans AP protects normal cells from toxic effects of Tamoxifen and helpsTamoxifen to kill cancer cells at half of its dose.11. Overall, we identified a molecule that has following advantageouscharacteristics: anticancer activity; anti-atherosclerosis activity; andutility as an adjunct therapy to reduce toxicity and increase efficacyof currently used chemotherapeutic drugs.

REFERENCES

-   1. Sagar, S.; Esau, L.; Moosa, B.; Khashab, N. M.; Bajic, V. B.;    Kaur, M. Cytotoxicity and apoptosis induced by a plumbagin    derivative in estrogen positive MCF-7 breast cancer cells.    Anti-cancer agents in medicinal chemistry 2014, 14, 170-180.-   2. Cruz, P. M.; Mo, H.; McConathy, W. J.; Sabnis, N.; Lacko, A. G.    The role of cholesterol metabolism and cholesterol transport in    carcinogenesis: a review of scientific findings, relevant to future    cancer therapeutics. Frontiers in pharmacology 2013, 4, 119.-   3. Gorin, A.; Gabitova, L.; Astsaturov, I. Regulation of cholesterol    biosynthesis and cancer signaling. Current opinion in pharmacology    2012, 12, 710-716.-   4. dos Santos, C. R.; Domingues, G.; Matias, I.; Matos, J.; Fonseca,    I.; de Almeida, J. M.; Dias, S. LDL-cholesterol signaling induces    breast cancer proliferation and invasion. Lipids in health and    disease 2014, 13, 16.-   5. Kaiser, J. Cancer. Cholesterol forges link between obesity and    breast cancer. Science (New York, N.Y.) 2013, 342, 1028.-   6. Llanos, A. A.; Makambi, K. H.; Tucker, C. A.; Wallington, S. F.;    Shields, P. G.; Adams-Campbell, L. L. Cholesterol, lipoproteins, and    breast cancer risk in African American women. Ethnicity & disease    2012, 22, 281-287.-   7. Janes, P. W.; Ley, S. C.; Magee, A. I. Aggregation of lipid rafts    accompanies signaling via the T cell antigen receptor. The Journal    of cell biology 1999, 147, 447-461.-   8. Merritt, E. A.; Sixma, T. K.; Kalk, K. H.; van Zanten, B. A.;    Hol, W. G. Galactose-binding site in Escherichia coli heat-labile    enterotoxin (LT) and cholera toxin (CT). Molecular microbiology    1994, 13, 745-753.-   9. Lewis, G. F.; Rader, D. J. New insights into the regulation of    HDL metabolism and reverse cholesterol transport. Circulation    research 2005, 96, 1221-1232.-   10. Hao, W.; Friedman, A. The LDL-HDL profile determines the risk of    atherosclerosis: a mathematical model. PloS one 2014, 9, e90497.-   11. Le Goff, W.; Guerin, M.; Chapman, M. J. Pharmacological    modulation of cholesteryl ester transfer protein, a new therapeutic    target in atherogenic dyslipidemia. Pharmacology & therapeutics    2004, 101, 17-38.-   12. Tchoua, U.; D'Souza, W.; Mukhamedova, N.; Blum, D.; Niesor, E.;    Mizrahi, J.; Maugeais, C.; Sviridov, D. The effect of cholesteryl    ester transfer protein overexpression and inhibition on reverse    cholesterol transport. Cardiovascular research 2008, 77, 732-739.-   13. Villard, E. F.; El Khoury, P.; Duchene, E.; Bonnefont-Rousselot,    D.; Clement, K.; Bruckert, E.; Bittar, R.; Le Goff, W.; Guerin, M.    Elevated CETP activity improves plasma cholesterol efflux capacity    from human macrophages in women. Arteriosclerosis, thrombosis, and    vascular biology 2012, 32, 2341-2349.-   14. Miller, N. E. CETP inhibitors and cardiovascular disease: Time    to think again. F1000Research 2014, 3, 124.-   15. Danilo, C.; Frank, P. G. Cholesterol and breast cancer    development. Current opinion in pharmacology 2012, 12, 677-682.-   16. Niendorf, A.; Nagele, H.; Gerding, D.; Meyer-Pannwitt, U.;    Gebhardt, A. Increased LDL receptor mRNA expression in colon cancer    is correlated with a rise in plasma cholesterol levels after    curative surgery. International journal of cancer. Journal    international du cancer 1995, 61, 461-464.-   17. Solomon, K. R.; Freeman, M. R. The complex interplay between    cholesterol and prostate malignancy. The Urologic clinics of North    America 2011, 38, 243-259.-   18. Nelson, E. R.; Wardell, S. E.; Jasper, J. S.; Park, S.;    Suchindran, S.; Howe, M. K.; Carver, N. J.; Pillai, R. V.;    Sullivan, P. M.; Sondhi, V.; Umetani, M.; Geradts, J.;    McDonnell, D. P. 27-Hydroxycholesterol links hypercholesterolemia    and breast cancer pathophysiology. Science (New York, N.Y.) 2013,    342, 1094-1098.-   19. Muntoni, S.; Atzori, L.; Mereu, R.; Satta, G.; Macis, M. D.;    Congia, M.; Tedde, A.; Desogus, A.; Muntoni, S. Serum lipoproteins    and cancer. Nutrition, metabolism, and cardiovascular diseases: NMCD    2009, 19, 218-225.-   20. Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.;    Lundback, T.; Nordlund, P.; Martinez Molina, D. The cellular thermal    shift assay for evaluating drug target interactions in cells. Nature    protocols 2014, 9, 2100-2122.

1. A method of treating a subject who suffers from a cancer, the methodcomprising administering to the subject a therapeutically effectiveamount of at least one Cholesteryl Ester Transfer Protein (CETP)inhibitor selected from the group consisting of rosuvastatin,rivastatin, pitavastatin, lovastatin, simvastatin, plumbagin andplumbagin analogues and derivatives including acetyl plumbain,pravastatin, fluvastatin, dalceptrpib, anacetrapib, evacetrapib,torcetrapib, atorvastatin (preferably atorvastatin hemi-calcium),cerivastatin, CETP inhibitors described in U.S. Pat. Nos. 7,652,049,6,140,343, 6,197,786, 6,723,752 (preferably(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol),and U.S. Pat. No. 5,512,548, CETP-inhibitory rosenonolactone derivativesand phosphate-containing analogs of cholesteryl ester described in J.Antibiot., 49(8): 815-816 (1996) and Bioorg. Med. Chem. Lett.;6:1951-1954 (1996), propanethioic acid, 2-methyl-, S-[2-[[[1-(2ethylbutyl)cyclohexyl]carbonyl]amino]phenyl]ester (Dalcetrapib),S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-3-phenylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]3-pyridinethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]chlorothioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]methoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]phenoxy-thio acetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclopropanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-4-carbamoylthiobutyrate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-hydroxy-2-methylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]thioacetate;S-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S[4,5-dichloro-2-(1-isopentylcyclopentanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;O-methyl S-[2-(1-isopentylcyclohexanecarbonylaminophenylmonothiocarbonate;S-[2-(1-methylcyclohexanecarbonylamino)phenyl]S-phenyldithiocarbonate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]N-phenylthiocarbamate;S-[2-(pivaloylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(2-cyclohexylpropionylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-pentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclohexylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopropylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcycloheptanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcyclobutanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-nitrophenyl]2,2-dimethylthiopropionate;S-[4-cyano-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[5-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-difluoro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[5-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;bis-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]disulfide;2-tetrahydrofurylmethyl-2-(1-isopentylcyclohexanecarbonylamino)phenyldisulfide; N-(2-mercaptophenyl)-1-ethylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-propylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-butylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-isobutylcyclohexanecarboxamide;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclohexanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiobenzoate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]5-carboxythiopentanoate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-methylphenyl]thioacetate;bis-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]disulfide;N-(2-mercaptophenyl)-1-(2-ethylbutyl)cyclohexanecarboxamide; S[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-methylthiopropionate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]-acetylpiperidine-4-thiocarboxylate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]thioacetate;S-[2-[1(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2,2-dimethylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]methoxythioacetate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-hydroxy-2-methylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]4-chlorophenoxythioacetate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;andS-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]-1-acetyl-piperidine-4-thiocarboxylateand analogs, derivatives, pharmaceutically acceptable salts,enantiomers, diastereomers, solvates and polymorphs thereof.
 2. Themethod of claim 1, wherein the Cholesteryl Ester Transfer Protein (CETP)inhibitor is selected from the group consisting of plumbagin andanalogues/derivatives, rosuvastatin, rivastatin, pitavastatin,lovastatin, simvastatin, pravastatin, fluvastatin, dalceptrpib,anacetrapib, evacetrapib, torcetrapib, atorvastatin (preferablyatorvastatin hemi-calcium), cerivastatin and analogs, derivatives,pharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof.
 3. The method of claim 1, wherein the cancer isselected from the group consisting of carcinomas (e.g., squamous-cellcarcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cellcarcinomas), particularly those of the bladder, bowel, breast, cervix,colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas,prostate, and stomach; leukemias; benign and malignant lymphomas,particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign andmalignant melanomas; myeloproliferative diseases; sarcomas, particularlyEwing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma,myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumorsof the central nervous system (e.g., gliomas, astrocytomas,oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas,ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors,meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas);germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer,cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicularcancer, thyroid cancer, astrocytoma, esophageal cancer, pancreaticcancer, stomach cancer, liver cancer, colon cancer, and melanoma); mixedtypes of neoplasias, particularly carcinosarcoma and Hodgkin's disease;and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas(Beers and Berkow (eds.), The Merck Manual of Diagnosis and Therapy,17.sup.th ed. (Whitehouse Station, N.J.: Merck Research Laboratories,1999) 973-74, 976, 986, 988,
 991. 4. The method of claim 1, wherein thesubject is co-administered one or more additional anticancer agentsselected from the group consisting of microtubule-stabilizing agents,microtubule-disruptor agents, alkylating agents, antimetabolites,epidophyllotoxins, antineoplastic enzymes, topoisomerase inhibitors,inhibitors of cell cycle progression, platinum coordination complexesincluding everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101,pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886),AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197,MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFRinhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, aPARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TKinhibitor; an anti-HGF antibody, a PI3 kinase inhibitors, an AKTinhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focaladhesion kinase inhibitor, a Map kinase (mek) inhibitor, a VEGF trapantibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402, lucanthone, LY 317615,neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311,romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat,etoposide, gemcitabine, doxorubicin, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258,);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(x) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine,mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate,mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, teniposide, testosterone,thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-freepaclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic-trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, and mixtures thereof.
 5. The methodof claim 1, wherein the subject suffers from breast cancer.
 6. Themethod of claim 5, wherein the subject is co-administered one or moreadditional anticancer agents selected from the group consisting oftamoxifen, paclitaxel and fluorouracil (5-FU).
 7. The method of claim 6,wherein the subject is co-administered plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone), acetyl plumbagin or an analog,derivative, pharmaceutically acceptable salt, enantiomer, diastereomer,solvate or polymorph thereof.
 8. The method of claim 7, wherein thesubject is co-administered a compound selected from the group consistingof plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a compound offormula (I) or formula (II), and the analogs, derivatives,pharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof:

wherein R¹ is H, substituted or unsubstituted cyano, halo, carboxyl, ornitro; R² is H, substituted or unsubstituted C₁-C₁₂alkyl, or O—R′; R³ isH, substituted or unsubstituted —N(R′)₂, or —O—R′; R⁴ is H, —O—R′, orC₁-C₆ alkyl; and each R, when present, independently, is H, substitutedor unsubstituted C₁-C₆ alkyl, or a hydrolyzable moiety, such as acyl ortrialkylsilyloxy.
 9. The method of claim 1, wherein the subject suffersfrom a form of refractory breast cancer.
 10. The method of claim 9,wherein the subject has developed an acquired anti-estrogen resistance.11. The method of claim 7, wherein the subject exhibits an intrinsicresistance to anti-estrogen and anti-HER2 therapies. 12.-52. (canceled)53. A pharmaceutical composition comprising: (a) an amount of one orCholesteryl Ester Transfer Protein (CETP) inhibitors that istherapeutically effective in the treatment of a cancer; (b) one or moreadditional anticancer agents; and (c) optionally, a pharmaceuticallyacceptable excipient.
 54. The pharmaceutical composition of claim 53,wherein the one or more additional anticancer agents are selected fromthe group consisting of tamoxifen, paclitaxel and fluorouracil (5-FU).55. The pharmaceutical composition of claim 53, wherein the CholesterylEster Transfer Protein (CETP) inhibitor is selected from the groupconsisting of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), acetylplumbagin (AP), rosuvastatin, rivastatin, pitavastatin, lovastatin,simvastatin, pravastatin, fluvastatin, dalceptrpib, anacetrapib,evacetrapib, torcetrapib, atorvastatin (preferably atorvastatinhemi-calcium), cerivastatin and analogs, derivatives, pharmaceuticallyacceptable salts, enantiomers, diastereomers, solvates and polymorphsthereof.
 56. The pharmaceutical composition of claim 53, wherein theCholesteryl Ester Transfer Protein (CETP) inhibitor is selected from thegroup consisting of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), acompound of formula (I) or formula (II), and the analogs, derivatives,pharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof:

wherein R¹ is H, substituted or unsubstituted C₁-C₁₂alkyl, cyano, halo,carboxyl, or nitro; R² is H, substituted or unsubstituted C₁-C₁₂alkyl,or O—R′; R³ is H, substituted or unsubstituted C₁-C₁₂alkyl, —N(R′)₂, or—O—R′; R⁴ is H, —O—R′, or C₁-C₆ alkyl; and each R′, when present,independently, is H, substituted or unsubstituted C₁-C₆ alkyl, or ahydrolyzable moiety, such as acyl or trialkylsilyloxy.
 57. Thepharmaceutical composition of claim 53, wherein the compositioncomprises: (a) acetyl plumbagin; (b) one or more additional anticanceragents selected from the group consisting of tamoxifen, paclitaxel andfluorouracil (5-FU); and (c) optionally, a pharmaceutically acceptableexcipient. 58.-60. (canceled)
 61. A method of treating a cancer which isidentified to overexpress Cholesteryl Ester Transfer Protein (CETP) in apatient in need comprising administering to said patient atherapeutically effective amount of a Cholesteryl Ester Transfer Protein(CETP) inhibitor.
 62. The method according to claim 61 wherein said CETPinhibitor is at least one compound selected from the group consisting ofplumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a compound of formula(I) or formula (II), and the analogs, derivatives, pharmaceuticallyacceptable salts, enantiomers, diastereomers, solvates and polymorphsthereof:

wherein R¹ is H, substituted or unsubstituted C₁-C₁₂alkyl, cyano, halo,carboxyl, or nitro; R² is H, substituted or unsubstituted C₁-C₁₂alkyl,or O—R′; R³ is H, substituted or unsubstituted C₁-C₁₂alkyl, —N(R′)₂, or—O—R′; R⁴ is H, —O—R′, or C₁-C₆ alkyl; and each R′, when present,independently, is H, substituted or unsubstituted C₁-C₆ alkyl, or ahydrolyzable moiety, such as acyl or trialkylsilyloxy, and optionally atleast one additional CETP inhibitor and/or an additional anticanceragent.
 63. The method according to claim 61 wherein said CETP inhibitoris at least one compound selected from the group consisting of plumbaginand plumbagin analogues and derivatives including acetyl plumbain,rosuvastatin, rivastatin, pitavastatin, lovastatin, simvastatin,pravastatin, fluvastatin, dalceptrpib, anacetrapib, evacetrapib,torcetrapib, atorvastatin (preferably atorvastatin hemi-calcium),cerivastatin, CETP inhibitors described in U.S. Pat. Nos. 7,652,049,6,140,343, 6,197,786, 6,723,752 (preferably(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-et-hoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol),and U.S. Pat. No. 5,512,548, CETP-inhibitory rosenonolactone derivativesand phosphate-containing analogs of cholesteryl ester described in J.Antibiot., 49(8): 815-816 (1996) and Bioorg. Med. Chem. Lett.;6:1951-1954 (1996), propanethioic acid, 2-methyl-, S-[2-[[[1-(2ethylbutyl)cyclohexyl]carbonyl]amino]phenyl]ester (Dalcetrapib),S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-3-phenylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]3-pyridinethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]chlorothioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]methoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]phenoxy-thioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclopropanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-acetylamino-4-carbamoylthiobutyrate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]2-hydroxy-2-methylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclopentanecarbonylamino)phenyl]thioacetate;S-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S[4,5-dichloro-2-(1-isopentylcyclopentanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;O-methyl S-[2-(1-isopentylcyclohexanecarbonylaminophenylmonothiocarbonate;S-[2-(1-methylcyclohexanecarbonylamino)phenyl]S-phenyldithiocarbonate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]N-phenylthiocarbamate;S-[2-(pivaloylamino)-4-trifluoromethylphenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(2-cyclohexylpropionylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-pentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclopropylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-cyclohexylmethylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopropylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcycloheptanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[4,5-dichloro-2-(1-isopentylcyclobutanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-nitrophenyl]2,2-dimethylthiopropionate;S-[4-cyano-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[5-chloro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;S-[4,5-difluoro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]2,2-dimethylthiopropionate;S-[5-fluoro-2-(1-isopentylcyclohexanecarbonylamino)phenyl]2,2-dimethylthiopropionate;bis-[4,5-dichloro-2-(1-isopentylcyclohexanecarbonylamino)-phenyl]disulfide;2-tetrahydrofurylmethyl-2-(1-isopentylcyclohexanecarbonylamino)phenyldisulfide; N-(2-mercaptophenyl)-1-ethylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-propylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-butylcyclohexanecarboxamide;N-(2-mercaptophenyl)-1-isobutylcyclohexanecarboxamide;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]cyclohexanethiocarboxylate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]thiobenzoate;S-[2-(1-isopentylcyclohexanecarbonylamino)phenyl]5-carboxythiopentanoate;S-[2-(1-isopentylcyclohexanecarbonylamino)-4-methylphenyl]thioacetate;bis-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]disulfide;N-(2-mercaptophenyl)-1-(2-ethylbutyl)cyclohexanecarboxamide; S[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-methylthiopropionate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]2-methylthiopropionate-;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]-acetylpiperidine-4-thiocarboxylate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]thioacetate;S-[2-[1(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2,2-dimethylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]methoxythioacetate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]2-hydroxy-2-methylthiopropionate;S-[2-[1-(2-ethylbutyl)cyclohexanecarbonylamino]phenyl]4-chlorophenoxythioacetate;S-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]4-chlorophenoxythioacetate;andS-[2-(1-isobutylcyclohexanecarbonylamino)phenyl]-1-acetyl-piperidine-4-thiocarboxylateand analogs, derivatives, pharmaceutically acceptable salts,enantiomers, diastereomers, solvates and polymorphs thereof.
 64. Themethod according to claim 61 wherein said CETP inhibitor(s) isadministered in combination with at least one additional anticanceragent. 65.-70. (canceled)